Use of mutated subtilisin protease in cosmetic products

ABSTRACT

A mutated subtilisin-type protease that bears at least one mutation in its amino-acid sequence that causes the positive charge to be reduced or the negative charge to be increased in the substrate binding region of the molecule is used in cosmetic products. Such proteases show a surprisingly low skin and mucous membrane irritating potential.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. § 371of international application PCT/EP96/03589 filed on Aug. 14, 1996, theinternational application not being published in English. Thisapplication also claims priority under 35 U.S.C. § 119 to DE 195 30816.6, filed on Aug. 23, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the use of mutated proteolytic enzymes with alow skin irritation potential, namely mutated subtilisin proteases, incosmetic products, more particularly body-cleansing and body-careformulations and oral hygiene formulations.

Enzymes, such as proteases, lipases, amylases and cellulases, have longbeen used in detergents and cleaners—essentially to support theirwashing and cleaning performance. Among these enzymes, proteases occupya position of paramount importance.

2. Discussion of the Related Art

Proteases are enzymes which catalyze the hydrolysis of peptide bonds inprotein and peptide substrates and of ester bonds in certain terminalesters. Subtilisins are a family of bacterial extracellular proteaseswith molecular weights of about 20,000 to 45,000 dalton which can beobtained from soil bacteria, for example Bacillus amyloliquefaciens.Subtilisins belong to the group of serine proteases which initiate thenucleophilic attack on the peptide (ester) bond through a serine residueat the active site. They are physically and chemically wellcharacterized enzymes. The three-dimensional structure of certainsubtilisins was elucidated in detail by X-ray diffractograms (C. Betzel,G. P. Pal and W Saenger, (1988) Eur. J. Biochem. 178, 155-171; R. Bott,M. Ultsch, A. Kossiakoff, T. Graycar, B. Kartz and S. Power, (1988) J.Biol Chem. 263, 7895-7906; D. W. Goddette, C. Paech, S. S. Yang, J. R.Mielenz, C. Bystroff, M. Wilke and R. J. Fletterick, (1992) J. Mol.Biol. 228, 580-595; D. W. Heinz, J. P. Priestle, J. Rahuel, K. S. Wilsonand M. G. Grütter (1991) J. Mol. Biol. 217, 353-371; J. Kraut (1977)Ann. Rev. Biochem. 46, 331-358; D. J. Neidhart and G. A. Petsko (1988)Protein Eng. 2, 271-276; A. V. Teplyakov, I. P. Kuranova, E. H.Harutyunyan, B. K Vainshtein, C. Frömmel, W.-E. Höhne und K. S. Wilson(1990) J. Mol. Biol. 214, 261-279).

Subtilisins are widely used in commercial products, for example inlaundry and dishwashing detergents and in contact lens cleaners,and—above all for research purposes—in synthetic organic chemistry. Onemember of the subtilisin family, namely a highly alkaline protease whichcan be used in surfactant-containing formulations, is described inInternational patent application WO 91/02792. This alkaline proteasefrom Bacillus lentus (Bacillus lentus alkaline protease; BLAP) can beobtained in commercially useful quantities from the strain Bacilluslicheniformis ATCC 53926 which carries an expression plasmid thatexpresses the BLAP gene under the control of the promoter of thealkaline protease of Bacillus licheniformis ATCC 53926. The crystalstructure of BLAP has been determined (D. W. Goddette et al. (1992) J.Mol. Biol. 228, 580-595; WO 92/21760) and the coordinates were depositedat the Brookhaven Protein Data Bank. If an optimal sequence homology ofBLAP (269 amino acids) is aligned with that of subtilisin BPN′ (275amino acids), the following pattern is obtained: the BLAP positions 1 to35, 36 to 54, 55 to 160 and 161 to 269 correspond to positions 1 to 35,37 to 55, 57 to 162 and 167 to 275, respectively, in subtilisin BPN′.Unless otherwise indicated, the numbering of the amino acids used in thepresent specification corresponds to that of BLAP.

The following nomenclature is used to describe the protease variantsemployed in the present invention: [original amino acid; position of theN terminus of the ripe enzyme; substituted amino acid]. For example, thereplacement of valine by isoleucine in position 4 in BLAP is designatedV4I. The list of the standard abbreviations for the typical amino acidsis given in Table 1.

TABLE 1 Abbreviations of the amino acids A = Ala = Alanine C = Cys =Cysteine D = Asp = Aspartic acid E = Glu = Glutamic acid F = Phe =Phenyl alanine G = Gly = Glycine H = His = Histidine I = Ile =Isoleucine K = Lys = Lysine L = Leu = Leucine M = Met = Methionine N =Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine R = Arg =Arginine S = Ser = Serine T = Thr = Threonine V = Val = Valine W = Trp =Tryptophan Y = Tyr = Tyrosine

Where several mutations occur within the same protein molecule, this ischaracterized through the sum of the individual mutations, such as forexample S3T+V4I+A188P+V193M+V199I.

Protection against thermal and chemical inactivation and improvement ofwashing and cleaning performance and dermatological compatibility areprimary functions if new proteases are to be developed for industrialand institutional applications. Several enzymes, including proteases ofthe subtilisin type, have been developed by random mutagenesis orsite-specific mutagenesis. They provide some indicators as to howimproved thermal and chemical stability can be rationally achieved (D.A. Estell, T. P. Graycar and J. A. Wells (1985) J. Biol. Chem. 260,6518-6521; M. Matsumura, W. J. Becktel, M. Levitt and B. W. Matthews(1989) Proc. Natl. Sci. US 86, 6562-6566; M. W. Pantoliano, M. Whitlow,J. F. Wood, M. L. Rollence, B. C. Finzel, G. L. Gilliland, T. L. Poulosand P. N. Bryan (1988) Biochemistry 27, 8311-8317; A. J. Russell and A.R. Fersht (1987) Nature 328, 496-500; R. J. Siezen, W. M. De Vos, J. A.M. Leunissen and B. W. Dijkstra (1991) Protein Eng. 4, 719-737; J. H.van Ee (1991) Chimicaoggi (7/8), 31-35; J. A. Wells and D. A. Estell(1988) Trends Biochem. Sci. 13, 291-297). By contrast, the modificationof enzymatic activity, particularly improving or optimizing the activityrate for certain substrates, is a far more complex problem. EP 0 260 105discloses the production of subtilisin-BPN′-mutants with modified ratiosof transesterification rate to hydrolysis rate and nucleophilicspecificities by modifying specific amino acid residues within 15 Å ofthe catalytic triad. A. J. Russell and A. R. Fersht (1987), J. Mol. Biol193, 803-813, describe the isolation of a subtilisin-BPN′-mutant (DO99S)which has a modification to the surface charge at a distance of 14 to 15Å from the active center. This substitution influences the pH dependenceof the catalytic reaction of the subtilisin. None of these publicationsteaches whether the modifications to the amino acids also produce achange in the dermatological compatibility of the enzymes. EP 0 130 756,EP 0 247 647 and U.S. Pat. No. 4,760,025 disclose a saturation mutationprocess in which at least one mutation is inserted into the subtilisinBPN′ at the amino acid residues (BPN′ numbering) Asp32, Asn 155, Tyr104,Met222, Gly166, His64, Ser221, Gly169, Glu156, Ser33, Phe189, Tyr217and/or Ala152. Mutated proteases which show improved oxidativestability, modified substrate specificity and/or modified pH activityare obtained using this procedure. The documents in question also teachthat mutations in the vicinity of the active center of the protease havethe most influence on activity. However, none of the documents inquestion discloses a process with which it is possible to predictwhether and which changes in the amino acid sequence improve thedermatological compatibility of proteases.

Most of the information on the catalytic activity of subtilisins hasbeen gathered in investigations into the hydrolysis of smallwell-defined peptide substrates. Hitherto, little has been known aboutinteractions with large protein substrates. This applies in particularto information on the washing performance of proteases when theirsubstrate is bound to a textile surface and the catalysis has to takeplace in the presence of substances which interact with the enzyme, suchas bleaching agents, surfactants and builders. In addition, nothing isknown of the interaction of proteases with the substances normallypresent in skin-care and hair-care products and in oral hygieneproducts.

EP 0 328 229 discloses the isolation and characterization of PB92subtilisin mutants with improved properties when used in detergents onthe basis of the results of washing tests. This document teaches thatbiochemical properties are not reliable parameters for predicting enzymeperformance in washing. Processes mentioned therein for selectingmutations comprise the substitution of amino acids by other amino acidsin the same category (polar, non-polar, aromatic, charged, aliphatic andneutral), the substitution of polar amino acids, such as asparagine andglutamine, by charged amino acids and the increase in the anioniccharacter of the protease at the mutation sites which do not belong tothe active centers. There is no mention of a process for identifyingwhich specific amino acids should be modified.

There are several patent applications which describe modifications ofsubtilisin enzymes for improving their mode of action in detergents, forexample International patent applications WO 91/00345 and WO 92/11357.

European patent application EP 0 571 049 discloses certain mutatedproteolytic enzymes. These enzymes are said to be at least 70%homologous with the amino acid sequence of the PB92 serine protease andto differ from the PB92 serine protease in at least one amino acid atpositions 99, 102, 116, 126, 127, 128, 130, 160, 203, 211 and/or 212.The mutated protease is produced by growing a host strain transformedwith an expression vector which contains a DNA sequence and which codesfor the desired mutated protease.

Hitherto unpublished International patent application WO 95/23221describes mutated proteases which, when added to detergents andcleaners, improve their effectiveness.

The problem addressed by the present invention was to provide mutatedproteases which would show improved dermatological compatibility bycomparison with the original protease and which would be suitable foruse in cosmetic products, particularly body-cleaning and body-careformulations and oral hygiene formulations.

DESCRIPTION OF THE INVENTION

It has surprisingly been found that the mutated proteases mentioned inWO 95/23221 in particular satisfy this requirement.

The present invention relates to the use of mutated subtilisin proteasein cosmetic products which is characterized in that the mutatedsubtilisin protease contains at least one mutation in its amino acidsequence which leads to a reduced positive charge or to an increasednegative charge in the vicinity of that region of the molecule which isbound to the substrate (“substrate-binding region”).

Cosmetic products in which the mutated protease may be used inaccordance with the invention include, in particular, body-cleaning andbody-care formulations and oral hygiene formulations, for example soapsin solid and liquid form, peeling cremes, skin cremes, soft cremes,nourishing cremes, sun protection cremes, night cremes, skin oils,skin-care lotions and body aerosols, deodorants, shaving cremes andshaving foams, hair shampoos, hair rinses and foam baths, mouthwashesand toothpastes.

Surprisingly, the mutated proteases to be used in accordance with theinvention show a low potential for irritating the skin and mucousmembrane and, accordingly, are eminently suitable for use in theproducts mentioned above.

The present invention also relates to the use of the proteases mentionedabove for the production of skin-care, hair-care and body-careformulations. To produce these formulations, the individual componentsare mixed in known manner. The formulations in question, particularlywhere they contain lipophilic substances, may be present both as“water-in-oil” and as “oil-in-water” emulsions and may contain othertypical auxiliaries and additives.

Besides the proteases used in accordance with the invention as essentialingredients, the formulations may contain in particular surfactants,such as anionic, nonionic, cationic, amphoteric and/or zwitterionicsurfactants.

Suitable auxiliaries and additives are, for example, emulsifiers, oilcomponents, fats and waxes, thickeners, superfatting agents, biogenicagents, film formers, fragrances, dyes, pearlescers, preservatives andpH regulators.

Typical oil components are such substances are paraffin oil, vegetableoils, fatty acid esters, silicone oils, dialkyl ethers, fatty alcoholsand Guerbet alcohols, squalane and 2-octyl dodecanol, while suitablefats and waxes are, for example, spermaceti, beeswax, montan wax,paraffin and cetostearyl alcohol.

Suitable emulsifiers are, for example, sorbitan esters, monoglycerides,polysorbates, polyethylene glycol mono/difatty acid esters, highlyethoxylated fatty acid esters and high molecular weight siliconecompounds, for example dimethyl polysiloxanes with an average molecularweight of 10,000 to 50,000.

The superfatting agents used include such substances as polyethoxylatedlanolin derivatives, dicytin derivatives and fatty acid alkanolamides,the fatty acid alkanolamides also serving as foam stabilizers.

Suitable thickeners are, for example, polysaccharides, more particularlyxanthan gum, guar, agar agar, alginates and tyloses, carboxymethylcellulose and hydroxyethyl cellulose, also relatively high molecularweight polyethylene glycol monoesters and diesters of fatty acids,polyacrylates, polyvinyl alcohol and polyvinyl pyrrolidone andelectrolytes, such as sodium chloride and ammonium chloride.

Biogenic agents in the context of the invention are, for example, plantextracts, protein hydrolyzates and vitamin complexes.

Typical film formers are, for example, polyvinyl pyrrolidone, vinylpyrrolidone/vinyl acetate copolymers, polymers of the acrylic acidseries, quaternary cellulose derivatives and similar compounds.

Suitable preservatives are, for example, formaldehyde solutions,p-hydroxybenzoic acid esters or sorbic acid.

Pearlescers may be selected, for example, from glycol distearic acidesters, such as ethylene glycol distearate, and from fatty acidmonoglycol esters.

Suitable dyes are any of the substances suitable and licensed forcosmetic purposes as listed, for example, in the publication entitled“Kosmetische Fäbemittel” of the Farbstoffkommission der deutschenForschungsgemeinschaft, published by Verlag Chemie, Weinheim, 1984.These dyes are normally used in concentrations of 0.001 to 0.1% byweight, based on the mixture as a whole.

Cremes in particular may contain—optionally in addition to the additivesalready mentioned—antioxidants such as, for example, butylhydroxytoluene and tocopherol, moisturizers such as, for example,glycerol, sorbitol, 2-pyrrolidine-5-carboxylate, dibutyl phthalate,gelatin, polyglycols with an average molecular weight of 200 to 600, pHbuffers such as, for example, the lactic acid/triethanolamine or lacticacid/NaOH system, mild surfactants such as, for example, alkyloligoglucosides, fatty alcohol ether sulfates, fatty acid isethionates,taurides and sarcosinates, ether carboxylic acids, sulfosuccinates,protein hydrolyzates and fatty acid condensates, sulfotriglycerides,short-chain glucamides, phospholipids, plant extracts, for exampleextracts of Aloe vera, sun protection agents such as, for example,ultrafine titanium dioxide or organic substances, such as p-aminobenzoicacid and esters thereof, ethylhexyl-p-methoxycinnamic acid ester,2-ethoxyethyl-p-methoxycinnamic acid ester, butyl methoxydibenzoylmethane and mixtures thereof and so-called active-substanceaccelerators, more particularly essential oils such as, for example,eucalyptus oil, menthol and the like.

In order to be able to use proteases in formulations of the typementioned above, they should produce only very slight, if any,irritation of the skin and mucous membranes.

The original protease type from which the mutated proteases to be usedin accordance with the invention are derived is preferably anabove-described alkaline Bacillus lentus protease (BLAP) which isobtained from the strain DSM 5483 and which has 269 amino acid units, amolecular weight of 26,823 dalton and a calculated isoelectric point of9.7, based on pK standard values. The BLAP gene can be obtained in knownmanner by isolation of the chromosomal DNA from the B. lentus strain DSM5483, preparation of DNA samples with a homology against the DNAsequences of the regions coding for the B. lentus protease, productionof genome libraries from the isolated chromosomal DNA and selection ofthe libraries for the interesting genes by hybridization of the samples.Mutants of the BLAP mentioned with improved stability to heat andsurfactants are described in International patent application WO92/21760.

It has now been found that a reduced skin and mucous membrane irritationpotential can be obtained by carrying out amino acid modificationswithin the substrate-binding region of the enzyme which lead to anincrease in the negative charge. According to the present invention,this can be done, for example, by increasing negatively charged aminoacid residues or reducing positively charged amino acids residues in thesubstrate-binding region within a radius of 7 Å, the substrate-bindingregion being locatable with the aid of a bound substrate molecule suchas, for example, MPF. In particular, amino acid modifications atpositions 99, 154 and 211 in the BLAP variants M130 and M131 of Bacilluslentus known from WO 94/21760 lead to a reduced skin and mucous membraneirritation potential. The mutant M130 contains four amino acidmodifications by comparison with native BLAP: S3T, A188P, V193M andV199I. The mutant M131 contains five amino acid modifications bycomparison with native BLAP: S3T, V4I, A188P, V193M and V199I. The aminoacid sequence for the protease M130 or M131 is reproduced in SEQ ID No.2 or SEQ ID No. 1 of International patent application WO 92/21760. M130and M131 may serve as a basis for further amino acid modifications forobtaining proteases with a reduced irritation potential. Preferredprotease mutants for the use according to the invention are thoseobtained by replacement of at least one amino acid of the proteases M130or M131 in which the amino acid is selected from the group consisting ofarginine at position 99, serine at position 154 and leucine at position211. These mutants and their production are disclosed in Internationalpatent application WO 95/23221 and are designated there as F11(S3T+R99S+A188P+V193M+V199I), F43 (S3T+V4I+R99G+A188P+V193M+V199I), F44(S3T+V4I+R99A+A188P+V193M+V199I), F45 (S3T+V4I+R99S+A188P+V193M+V199I),F46 (S3T+V4I+S154E+A188P+V193M+V199I), F47(S3T+V4I+S154D+A188P+V193M+V199I), F49(S3T+V4I+A188P+V193M+V199I+L211D), F54(S3T+V4I+R99G+A188P+V193+V199I+L211D) and F55(S3T+V4I+S154D+A188P+V193M+V199I+L211D ). The proteolytic activity maybe measured as follows using the method described in Tenside 7 (1970)125, i.e. by discontinuous determination using casein as substrate: theconcentrations of the substrate solution are 12 mg per ml casein(prepared in accordance with Hammarsten; supplier: Merck, Darmstadt, No.2242) and 30 mM tris in synthetic tap water (aqueous solution of 0.029%(weight/v) CaCl₂. 2H₂O, 0.014% (weight/v) MgCl₂. 6H₂O and 0.021%(weight/v) NaHCO₃) with a hardness of 15°dH (German hardness). Thesubstrate solution is heated to 70° C. and the pH is adjusted to 8.5with 0.1 N NaOH at 50° C. The protease solution is prepared with 2%(weight/v) of anhydrous pentasodium tripolyphosphate in synthetic tapwater, the pH being adjusted to 8.5 with hydrochloric acid. 200 μl ofthe enzyme solution are added to 600 μl of the casein substratesolution. The mixture is incubated for 15 minutes at 50° C. The reactionis terminated by addition of 600 μl of 0.44 M trichloroacetic acid (TCA)and 0.22 M sodium acetate in 3% (VN) acetic acid. After cooling on icefor 15 minutes, the TCA-insoluble protein is removed by centrifugation,an aliquot of 900 μl is mixed with 300 μl of 2 N NaOH and the extinctionof the resulting mixture which contains TCA-soluble peptides is measuredat 290 nm. Control values are obtained by adding 600 μl of the TCAsolution to 600 μl of casein solution followed by 200 μl of enzymesolution. By definition a protease solution which produces a change inextinction of 0.550 OD at 290 nm under the conditions of this test hasan activity of 10 PU per ml.

EXAMPLES Example 1

Determination of the Irritation Potential

The irritation potential of proteases to be used in accordance with theinvention was investigated using F49 and—for comparison—BLAP asexamples. It was determined on guinea pigs in the course of the BuehlerTest for dose determination (E. V. Buehler, Arch. Dermatol. 1965, 91,171-177). To this end, a 10% solution of the protease was topicallyapplied to the shaved skin and fixed for 6 hours by means of a bandage(Scotchpak® Non-Woven Patch, a product of Minnesota Mining andManufacturing Company). The areas of skin of interest were evaluated 24,48 and 72 hours after removal of the bandages.

These tests were carried out on four guinea pigs (strain: “Pirbrightwhite”), the solutions being applied in at least two places per animal.The results are set out in Tables 2 and 3 below and represent meanvalues of erythema and odema formation. The results show that themutated protease to be used in accordance with the invention has a farlower irritation potential than the unmodified protease BLAP.

In the Tables, the figures 0, 1 and 2 stand for the number of areas ofskin on an animal where skin irritation was observed, s stands forslight crust formation at the margins of the treated skin areas and astands for swelling.

TABLE 2 Epidermal application of F49 Skin evaluation after removal ofbandage Animal Body weight Concentration Activity After 24 h After 48 hAfter 72 h Body weight No. on day - 5 (g) (w/w) (PU/ml) Erythema OdemaErythema Odema Erythema Odema on day - 7 (g) 5 347 10.52 20000 1 0 2a 22a 2 359 5.26 10000 1 0 1 0 1a 0 6 391 10.52 20000 1 0 1 2 1a 2 403 5.2610000 1 0 1 0 1a 0 7 400 2.63 5000 1 0 1 0 1 0 416 1.31 2500 0 0 0 0 0 00.52 1000 0 0 0 0 0 0 0.26 500 0 0 0 0 0 0 8 379 2.63 5000 1 0 1 0 1a 0393 1.31 2500 0 0 0 0 0 0 0.52 1000 0 0 0 0 0 0 0.26 500 0 0 0 0 0 0Observed systemic symptoms: none

TABLE 3 Epidermal application of BLAP (comparison) Skin evaluation afterremoval of bandage Animal Body weight Concentration Activity After 24 hAfter 48 h After 72 h Body weight No. on day - 5 (g) (w/w) (PU/ml)Erythema Odema Erythema Odema Erythema Odema on day - 7 (g) 1 351 8.6020000 1a 0 2as 2 2as 2 355 4.30 10000 1 0 1 0 1s 0 2 325 8.60 20000 1a 02as 2 2as 2 345 4.30 10000 1 0 1 1 1s 0 3 372 2.15 5000 1 0 2 1 2s 2 3681.07 2500 1 0 0 0 0 0 0.42 1000 0 0 0 0 0 0 0.21 500 0 0 0 0 0 0 4 3622.15 5000 1 0 2 2 2s 1 377 1.07 2500 0 0 1 0 1s 0 0.42 1000 0 0 1 0 1 00.21 500 0 0 0 0 0 0 Observed systemic symptoms: none

What is claimed is:
 1. A cosmetic composition comprising: (a) at leastone mutant Bacillus lentus alkaline protease, wherein the mutantprotease comprises a Bacillus lentus alkaline protease having one ormore amino acid substitutions in its amino acid sequence, wherein atleast one of the substitutions is at a position selected from 99, 154,or 211 or combinations thereof relative to the unmodified Bacilluslentus alkaline protease, leads to a reduced positive charge or anincreased negative charge, and results in the mutant protease havingreduced tissue surface irritation relative to the modified or unmodifiedBacillus lentus alkaline protease not having these substitutions; and(b) one or more cosmetic materials compatible with said mutant protease.2. The cosmetic composition of claim 1, wherein the mutant protease is aprotease M130, or a protease M131, or both a protease M130 and aprotease M131, and wherein said mutant protease comprises one or moreamino acid substitutions at a position selected from 99, 154, or 211, orcombinations thereof.
 3. A cosmetic composition comprising (a) at leastone mutant Bacillus lentus alkaline protease, wherein the mutantprotease is a protease M130 or a protease M131 modified by one or moreamino acid substitutions, wherein at least one of the amino acidsubstitutions is at a position selected from 99, 154, or 211 orcombinations thereof relative to the unmodified Bacillus lentus alkalineprotease from which the M130 protease or M131 protease is obtained,wherein the substitution at a position selected from 99, 154, or 211 orcombinations thereof leads to a reduced positive charge or an increasednegative charge, and results in the mutant protease having reducedtissue surface irritation relative to the M130 protease or M131 proteasenot having this substitution; and (b) one or more cosmetic materialscompatible with said mutant protease.
 4. The cosmetic composition ofclaim 3, wherein the mutant protease has an amino acid substitution atposition 99 relative to the unmodified Bacillus lentus alkaline proteaseand the substituent amino acid is selected from glycine, alagite, orserine.
 5. The cosmetic composition of claim 3, wherein the mutantprotease has an amino acid substitution at position 154 relative to theunmodified Bacillus lentus alkaline protease and the substituent aminoacid is glutanic acid or aspartic acid.
 6. The cosmetic composition ofclaim 3, wherein the mutant protease as an amino acid substitution atposition 211 relative to the unmodified Bacillus lentus alkalineprotease and the substituent amino acid is glutamic acid or asparticacid.
 7. The cosmetic composition of claim 3, wherein the mutantprotease has one or more mutations selected from the group consisting ofR99G, R99S, R99A, L211D), L211E, S154D, and S154E.
 8. A method ofavoiding or reducing tissue surface irritation due to contact with aprotease containing cosmetic composition, comprising contacting a tissuesurface with a cosmetic composition comprising at least one mutantBacillus lentus alkaline protease, wherein the mutant protease comprisesa Bacillus lentus alkaline protease having one or more no acidsubstitutions in its amino acid sequence, wherein at least one of thesubstitutions (i) occurs at a position lying within a 7 Å radius of thesubstrate binding region of the unmodified Bacillus lentus alkalineprotease, (ii) leads to a reduced positive charge or an increasednegative charge and (iii) results in the mutant protease having reducedtissue surface irritation relative to the modified or unmodifiedBacillus lentus alkaline protease not having these substitutions withinthe 7 Å radius.
 9. The method of claim 8, wherein at least one of thesubstitutions within the 7 Å radius of the substrate binding region thatleads to the reduced positive charge or increased negative charge is ata position selected from 99, 154, or 211 or combinations thereof,relative to the unmodified Bacillus lentus alkaline protease.
 10. Themethod of claim 9, wherein the mutant protease has one or moresubstitutions selected from the group consisting of R99G, R99S, R99A,L211D, L211E, S154D, and S154E.
 11. The method of claim 8 wherein themutant protease is a protease M130, or a protease M131, or both aprotease M130 and a protease M131, and wherein said mutant protease ismodified by one or more amino acid substitutions in its amino acidsequence at a position that lies within a 7 Å radius of the substratebinding region of the unmodified Bacillus lentus alkaline protease,wherein said one or more amino acid substitutions leads to a reducedpositive charge or an increased negative charge.
 12. The method of claim11, wherein the mutant protease has one or more amino acid substitutionsat a position selected from 99, 154, or 211 or combinations thereofrelative to the unmodified Bacillus lentus alkaline protease.
 13. Amethod of preparing a cosmetic composition, comprising mixing at leastone mutant Bacillus lentus alkaline protease with at least one cosmeticmaterial compatible with the mutant protease, wherein the mutantprotease comprises a Bacillus lentus alkaline protease having one ormore amino acid substitutions in its amino acid sequence, wherein atleast one of the substitutions (i) occurs at a position lying within a 7Å radius of the substrate binding region of the unmodified Bacilluslentus alkaline protease, (ii) leads to a reduced positive charge or anincreased negative charge and (iii) results in the mutant proteasehaving reduced tissue surface irritation relative to the modified orunmodified Bacillus lentus alkaline protease not having thesesubstitutions within the 7 Å radius.
 14. The method of claim 13 whereinat least one of the amino substitutions within the 7 Å radius of thesubstrate binding region that leads to the reduced positive charge orincreased negative charge is at a position selected from 99, 154, or 211or combinations thereof, relative to the unmodified Bacillus lentusalkaline protease.
 15. The method of claim 14, wherein the mutantprotease has one or more substitutions selected from the groupconsisting of R99G, R99S, R99A, L211D, L211E, S154D, and S154E.
 16. Themethod of claim 13, wherein the mutant protease is a protease M130, or aprotease M131, or both a protease M130 and a protease M131, and whereinsaid mutant protease is modified by one or more amino acid substitutionsin its amino acid sequence at a position that lies within a 7 Å radiusof the substrate binding region of the unmodified Bacillus lentusalkaline protease, wherein said one or more amino acid substitutionsleads to a reduced positive charge or an increased negative charge. 17.The method of claim 16, wherein the mutant protease has one or moresubstitutions at a position selected from 99, 154, or 211 orcombinations thereof relative to the unmodified Bacillus lentus alkalineprotease.